ANALYTICAL
97, 248-254
BIOCHEMISTRY
Chemiluminescence
HIDETOSHI School
of Pharmaceutical
(1979)
Enzyme Immunoassay Peroxidase as Label ARAKAWA, Sciences,
Showa
MASAKO University,
MAEDA, Hatanodai,
of Cortisol
AND AKIO Shinagawa-ku.
Using
TSUJI Tokyo,
Japan
Received December 5, 1978 A new and highly sensitive enzyme immunoassay of cortisol was established using horseradish peroxidase as the label. Separation of free and bound cortisol was effected by insolubilized anti-cortisol antibody which was prepared by coupling the purified immunoglobulin G of antiserum with Sepharose 4B. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. Comparison of assay results obtained by radioimmunoassay and this enzyme immunoassay showed excellent agreement of results in all cases (r = 0.913). The detection limit of cortisol was about 1Opgper assay tube. This enzyme immunoassay,is applicable to the routine determination of plasma cortisol.
Since the first report by Van Weemen and Schuurs (l), a number of studies (2,3) have been reported on the enzyme immunoassay (EIA)’ for various hormones and drugs. EIAs, like radioimmunoassay (RIA), make use of conjugate to amplify the sensitivity of immunological detection methods. However, many of them are not as sensitive as the corresponding RIAs, because enzyme activities in EIAs are usually assayed colorimetrically. Fluorophotometric methods for the determination of enzyme activity have been employed in order to increase the sensitivities of EIAs using glucoamylase (4), p-D-galactosidase (5,6), and peroxidase (7,8). In this report, we have attempted to develop a highly sensitive and clinically useful EIA for cortisol in plasma using peroxidase as a label enzyme, based on chemiluminescence reaction. MATERIALS Reagents. Luminol, oxide, S-aminosalicylic
AND METHODS
30% hydrogen acid, tyramine,
perand
1 Abbreviations used: EIA, enzyme immunoassay; RIA, radioimmunoassay; BSA, bovine serum albumin: IgG, immunoglobulin G; HRP, horseradish peroxidase. 0003-2697/79/120248-07$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
248
cyanogen bromide were purchased from Tokyo Kasei Company (Tokyo). Sepharose 4B, Bio-Gel P 60, and DEAE-cellulose were products of Seikagakukogyo Company (Tokyo). Cortisol and various steroids were obtained from Sigma Chemical Company, and horseradish peroxidase (types III and VI) used as the label enzyme was also purchased from Sigma Chemical Company. Other reagents were of reagent grade. 6 /3Hemisuccinoxycortisol, cortisol 3-( Ocarboxymethyl)oxime, and antiserum (rabbit) against 6 rY-hemisuccinoxycortisolBSA were supplied from Dr. D. K. Fukushima, the Institute for Steroid Research and the Division of Oncology, Montefiore Hospital and Medical Center, New York. 21-Hemisuccinoxycortisol and cortisol 3-( 0-carboxymethyl)oxime were prepared according to the literature (9). The anti-cortisol sera against their BSA conjugates were prepared by the Central Research Institute of Tori Pharmaceutical Company and the Research Division of Teikokuzoki Company (Tokyo). The IgG fraction of antiserum was purified by DEAEcellulose column chromatography after precipitation with ammonium sulfate.
CHEMILUMINESCENCE
ENZYME
IMMUNOASSAY
OF CORTISOL
249
lene chloride for 1 min. A 50-~1 aliquot of antibody (imthe organic phase was transferred to a glass The insolubilized antibody of cortisol was prepared by the method of tube and dried under a stream of nitrogen, Afen et al. (10). Sepharose 4B (50 ml) was and the residue was dissolved by adding activated at pH 10.5 by mixing with 50 ml 0.1 ml of phosphate buffer containing 0.1% BSA. The resultant sample solution was subof a cyanogen bromide solution (50 mg/ml aqueous solution) for 7 min. After washing mitted to the enzyme immunoassay. with water and 0.1 M sodium bicarbonate solution, the activated Sepharose 4B was Enzyme Immunoassay Procedure mixed with 0.1 g purified anti-cortisol IgG (rabbit) in 10 ml of 0.1 M sodium bicarbonate Method A (jirst-antibody solid-phase of a sample solution. The reaction was run at room tem- method). One-tenth milliliter perature overnight with slow stirring. Then, solution or a cortisol standard solution conthe coupled product was washed by the taining 0 to 1 ng of cortisol was incubated method of Wide (11). The washed insoluovernight at 4°C with 0.5 ml of a diluted imsuspension (1:50) (insolubilized antibody was finally suspended in munosorbent bilized first antibody) on a immunorotor. buffer and stored at 4°C. The insolubilized second antibody was also prepared by the After addition of 0.5 ml of a diluted HRPsame procedure with anti-rabbit IgG frac- cortisol conjugate solution (1:l X 104-3 x 104), the mixture was again incubated for tion (goat). Preparation oj’ HRP-cortisol conjugate. 4 h at 4°C on a immunorotor. The incubation HRP-Cortisol conjugates were prepared by mixture was centrifuged at 3000 t-pm for 5 the mixed anhydride method from 2 l-hemimin. One milliliter of the supernatant was succinoxycortisol, cortisol 3-( O-carboxyaspirated off, and the precipitated immunosorbent in the tube was washed three times methyl)oxime, and 6-p-hemisuccinoxycortieach with 2 ml of saline. And then, the persol. For example, 1 mg of 21-hemisuccinoxyoxidase activity was measured by the chemicortisol was dissolved in 0.2 ml of dioxane, and then 10 ,ul of tri-n-butylamine and 4 ~1 luminescence (12), fluorophotometric (13), (1) method. of isobutylchlorocarbonate were added to it. or spectrophotometric Method B (double-antibody solid-phase The resultant solution was stirred for 30 min at 10°C and then mixed with cooled method). Each 0.1 ml of a diluted anti-cortisol 1 ml enzyme solution containing 15 mg of serum (1:500) and a cortisol standard soluhorseradish peroxidase. The mixture was tion or a sample solution was mixed and kept at pH 8.0-8.5 for 5 h in ice-water bath. incubated for 1 hat room temperature. After The resultant reaction mixture was dialyzed addition of 0.5 ml of a diluted insolubilized against 0.05 M phosphate buffer and then second-antibody suspension ( 1:50), the chromatographed on Bio-Gel P-60 column mixture was incubated overnight at 4°C equilibrated in 0.05 M phosphate buffer con- on a immunorotor. One-half milliliter of a taining 0.9% sodium chloride (pH 7.0). The HRP-cortisol conjugate solution (1: 1. x 104excluded peak fractions were diluted with 3 x 104) was then added to all tubes which 0.05 M phosphate buffer containing 0.1% were incubated for a further 1 h at 4°C. The BSA, and stored at 4°C. 6-@Hemisuccinoxyincubation mixture was centrifuged at 3000 cortisol and cortisol 3-( O-carboxymethyl)rpm for 5 min, and the supernatant was oxime were also conjugated with HRP by aspirated and discarded. The immunothe same procedure as described above. sorbent was washed three times each with Plasma extraction. Ten microliters of 2 ml of saline, and then submitted to plasma was diluted with 50 ~1 of distilled measurement of the peroxidase activity water, and extracted with 500 ~1 of methyby the chemiluminescence method.
Preparation munosorbent).
of insolubilized
250
ARAKAWA,
MAEDA,
Method C (double-antibody method). The assay was caried out in a similar manner to the method described by Van Weemen et al. (1).
AND TSUJI
methods. In this paper, HRP was selected for the label enzyme, because HRP is widely used in various EIAs and is one of the best studied enzymes, available cheaply, and very stable under assay and storage conditions. Several procedures for coupling enzyme to hormone or drug have been developed. In the case of steroids, the mixed anhydride method (17) and soluble carbodiimide method were used for preparation of conjugate. Both methods were examined in this paper, and the better conjugate was obtained by the mixed anhydride method. The highest active fraction obtained by a gel chromatographic purification could be used for the assay in 1 x 104- to 3 x 104fold dilution. The binding ratio of cortisol to HRP in the conjugate was determined to be about 2 to 1 by spectrophotometry. The total enzyme activity of fractions that could be used in EIA was approximately 43% of the initial total activity. The choice of the separation procedure is important to enhance the sensitivity in heterogeneous EIA. First-antibody solidphase method (method A), double-antibody solid-phase method (method B), and doubleantibody method (method C) were ex-
Measurement of peroxidase activity by chemiluminescence method (12). Each 0.5
ml of 0.01 M phosphate buffer (pH 7.0), 0.01 M H,Oz solution, and 0.01 M luminol solution was added to the washed immunosorbent in the test tube, respectively. After mixing well by a Vortex-type mixer for 5 s, the chemiluminescence intensity was measured for 10 s by a photon counter (HTV-C 767, Hamamatsu TV Co., Japan). RESULTS AND DISCUSSION
Although radioimmunoassay methods have been widely used for determination of steroid hormones in biological fluids, EIAs of steroids have recently been developed, because of the biohazard and problems of disposal resulting from the use of radioisotopes. EIAs of cortisol using /3-D-galactosidase (14) and alkaline phosphatase (15,16) as label enzyme have been reported. In those EIAs, the enzyme activities were assayed by calorimetric 100
100
iR
Y 5
5 x
X 50 2 t L*
Ino 2
4 ’ 0.01
0.025
I 0.05
I 0.10 cortiso1
I 0.25
I 0.50
I 1.0
L &tube
2
5o
I h
1,
' 0.25
1
I
I
0.5
1.0
1
2.5
I
I
5.0
10
n&tube
cortiso1
FIG. 1. Standard curves of cortisol by chemiluminescence enzyme immunoassay. (A) (0 --) First-antibody solid-phase method (method A). (- - 0 - -) Double-antibody solid-phase method (method B). (II, andB) Chemiluminescence intensities of HRP-cortisol conjugate bound to immunosorbent in the absence and presence of cortisol, respectively. (B) Double-antibody method (method C). (F, and F) Chemiluminescence intensities of the supernatants in the absence and presence of cortisol, respectively. (T) Chemiluminescence intensity of the supernatant in the absence of anti-cortisol serum and cortisol.
CHEMILUMINESCENCE
ENZYME
amined. The typical standard curves by these methods are shown in Fig. 1. Fig. 1A was obtained by plotting (BIB,,) x 100 against the amount of cortisol (B, and B: the chemiluminescence intensities by HRPcortisol conjugate bound to immunosorbent in the absence and the presence of cortisol, respectively). Fig. 1B was prepared by plotting (F-F,)I( T-F,,) x 100 against the amount of cortisol (F, and F: the chemiluminescence intensities of the supernatants in the absence and the presence of cortisol, respectively. T: the chemiluminescence intensity of the supernatant in the absence of anti-cortisol serum and cortisol). Van Weemen et al. (1) and Avrameas et al. (18) showed that the double-antibody solidphase method gave higher sensitivity, greater precision, and more rapid separation than the insolubilized first-antibody method in EIAs for human chorionic gonadotropin and IgG, respectively. However, in our study, the sensitivity of the double-antibody solid-phase method is almost same as that of the insolubilized first-antibody method. Therefore, the first-antibody solidphase prepared from anti-cortisol serum against 21-hemisuccinoxycortisol-BSA was used for the separation of bound and free fraction in the assay of plasma samples (method A) and the double-antibody solidphase method (method B) was used for the test of comparison of heterologous and homologous combinations of antibodies and HRP-cortisol conjugates. As shown in Fig. lA, cortisol could be determined in the range from 10 pg to 2.5 ng. The sensitivity of these methods are comparable to that of the radioimmunoassay. The repeatability, assessed by evaluating the coefficients of variation at three cortisol levels (SO, 100 and 500 pg), were 6.6, 7.5, and 10.4% with method A and 8.2, 3.9, and 7.8% with method B, respectively. The cross-reactivity of some steroids with this method was examined. Crossreactivity of 1 l-desoxycortisol, 17+hydroxyprogesterone, corticosterone, progester-
IMMUNOASSAY
OF CORTISOL
251
one, and dehydroepiandrosterone were 83.7,38.5, 18.9,7.1, and0.9%, respectively. These values were approximately equal to those obtained by RIA using the same anticortisol serum which was used in EIA. The percentage of cross-reaction of 1I-desoxycortisol and 17 a-hydroxyprogesterone was considerably high, but the concentrations of these steroids in plasma are insignificant relative to that of cortisol. Therefore, any separation step prior to EIA was unnecessary for the assay of human plasma samples. The label enzyme activity is usually measured by visible or ultraviolet spectrophotometry. Fluorophotometric methods have been used in highly sensitive assays for the labels, glucoamylase (4), and /3-D-galactosidase (5,6). The peroxidase activity in EIA for estradiol has been assayed by fluorophotometric method (8) using homovanillic acid and H,O, as substrate. Recently, chemiluminescence reaction of luminol is used for the determination of certain trace metals and other substances of biological interest (19). Schroeder et al. have developed the specific protein-binding reaction monitored using bioluminescence reaction with ligand-NAD (20) or ligand-ATP conjugate (21). Puget et al. (22) have also reported the chemiluminescence technique for the determination of peroxidase and its application to the estimation of antibody bound to mouse lympocytes using luminolH,O, or pholad luciferin as substrate. In our previous paper, we established the assay of peroxidase activity using luminolHzOz as substrate and preliminarily applied it to EIA (12). This method could be favorably applied to the EIA of cortisol. The detection limit of cortisol was approximately 10 pg per assay tube, as shown in Fig. IA, comparable to that of RIA and superior to those of the EIAs for cortisol reported by Comoglio (15), Ogihara (16), and Kobayashi (18). The simple comparisons between sensitivities of assays are difficult, because they are dependent on various factors, such as the affinity of antigen-antibody reaction
252
ARAKAWA,
MAEDA,
and the separation methods of bound and free fractions. Therefore, the comparison between chemiluminescence and spectrophotometric and fluorophotometric methods for the assay of enzyme activity was carried out under the same immune reaction conditions. As shown in Table 1, the chemiluminescence method was more sensitive than the spectrophotometric method with 5-aminosalicylic acid and the fluorophotometric method with tyramine. Moreover, the time for the assay of enzyme activity by the chemiluminescence method was only 10 s. On the other hand, it took 30 min or more to get the measurable optical intensity by spectrophotometry and fluorophotometry. In EIAs of steroid, the systems in which the same steroid derivative is used both for preparing the immunogen and for preparing the steroid-enzyme conjugate are referred to as “homologous” in contrast to “heterologous” systems, in which different steroid derivatives are used. Van Weemen et al. (23) suggested that the sensitivity of EIA for estrogen could be considerably improved when the heterologous systems were used. Recently, Numazawa et al. (8) reported a similar effect in the EIA of estradiol, while Ogihara et al. (16) reported that cortisol could not be assayed TABLE COMPARISON OF RESULTS SPECTROPHOTOMETRIC
Method Fluorophotometric
method
OBTAINED METHODS
AND TSUJI
by the heterologous system using 21-hemisuccinoxycortisol-alkaline phosphatase conjugate and anti-cortisol antiserum raised against cortisol3-(O-carboxymethyboximeBSA conjugate. In this paper, the homologous and heterologous systems were brought about by utilizing the different positions on cortisol nucleus, namely C-3, C-6, and C-2 1, through which enzyme and carrier protein were linked to cortisol, respectively. The concentrations of cortisol which replaced 50% of HRP-cortisol conjugate bound to insolubilized antibody by method B were determined as the sensitivities of various systems. The results are given in Table 2. As shown in Table 2, the highest sensitivity was obtained by the heterologous system combining anti-6a+hemisuccinoxycortisol-BSA with HRP conjugate of cortisol 3-( 0-carboxymethyl)oxime, of which the midpoint is 0.12 &assay tube. However, the detection limit obtained by this system was almost the same as that of the homologous system with 21-hemisuccinoxycortisol. The steepness of the assay curves varied from each other. From these results, it is difficult to reach a systematic conclusion on the effect of the combination of antibody and HRPcortisol conjugate being used to the sensitivity of EIAs for cortisol, because the 1
BY CHEMILUMINESCENCE, FOR DETERMINATION
FLUOROPHOTOMETIUC OF PEROXIDASE ACTIVITY~
Added
AND
Sensitivity
Reproducibility
(PP)
(CV%)b
25
2.8-6.8
100 250
100.6 89.2
bzhl)
Recovery (%I
Spectrophotometric
method
100
2.9-11.6
250 500
111.6 105.8
Chemiluminescence
method
10
5.2-9.3
250 500
85.2 83.2
a Method A of the enzyme immunoassay was used. “n=5.
CHEMILUMINESCENCE
ENZYME
IMMUNOASSAY
TABLE MIDPOINTS
2
FOR CORTISOL IN ENZYME IMMUNOASSAY COMPARED WITH CORRESPONDING
Cortisol derivative in immunogen
(METHOD HOMOLOGOUS
B) WITH SITE HETEROLOGY ASSAYS”
6ol-Hemisuccinoxy cortisol-BSA
2 1-Hemisuccinoxy cortisol-BSA
Cortisol derivative in HRP conjugate
253
OF CORTISOL
Dilution of antibody
Cortisol 3-(O-carboxymethyl) oxime-BSA
Dilution of antibody
Midpoint
21-Hemisuccinoxy cortisol-HRP
500
0.4 ng (X 10000)
10
50 ng (X 1000)
8ooo
0.5 ng (X 10ooo)
6@Hemisuccinoxy cortisol-HRP
50
50 ng ( x 2500)
500
0.5 ng ( x 10000)
500
>l.O /.&g ( x 5000)
Cortisol 3-(O-carboxymethyl) oxime-HRP
20
>l.O /.&g ( x 5000)
1000
0.12 ng (X30000)
500
>I.0 pg ( x 5000)
n Value in parentheses: dilution of HRP-cortisol pension used in this experiment: 50 fold.
Dilution of antibody
conjugate. Dilution of insolubilized
immune reactivity of antibody and cortisolenzyme conjugate have large individual differences. The cortisol levels in 28 plasma samples were determined by method A of EIA and RIA in order to assess the reliability of the present method. As illustrated in Fig. 2, the values obtained by EIA and RIA are in excellent agreement, the correlation co-
I
Midpoint
I
1
50
100
Midpoint
second-antibody sus-
efficient for cortisol samples in the range from 5 to 250 rig/ml of plasma being 0.913. The values obtained by EIA tended to be slightly higher than the corresponding values obtained by RIA: In conclusion, a highly sensitive and practically useful EIA for cortisol in plasma has been established. The chemiluminescence reaction using peroxidase-luminol-H,O,
I
Cortisol
(g/ml
Enzyme
hWlOaSSay
I
150 in plasma) (Method
200
250
A)
FIG. 2. Correlation between EIA and RIA values of cortisol in plasma.
254
ARAKAWA,
MAEDA,
system was most sensitive and rapid for the assay of enzyme activity. This system may be applicable extensively for the determination of various steroid hormones and drugs in the biological materials.
9. Tsuji, A., Smulowtiz, M., Liang, J. S. C., and Fukushima, D. K. (1974) Steroids 24,739-751. 10. Afne, R.. Porath, J., and Emback, S. (1967) Nature (London) 214, 1302- 1304. 11. Wide, L. (1970) Acta Endocrinol. (Suppl.) 142, 207-218. 12.
ACKNOWLEDGMENTS This work was supported in part by a Grant 357620 from the Ministry of Education of Japan. The authors are indebted to Dr. David K. Fukushima (the Institute for Steroid Research and the Division of Oncology, Montefiore Hospital and Medical Center, New York) for a gift of anti-cortisol serum and cortisol derivatives, and Mr. H. Tanaka (the Central Research Institute of Tori Pharmaceutical Co., Tokyo) for preparing anti-corns01 serum.
13. 14. IS. 16. 17.
REFERENCES 1. Van Weemen, B. K., and Schuurs, A. H. W. M. (1971) FEBS Let?. 15, 232-236. 2. Wisdom, G. B. (1976) C/in. C&m. 22, 1243- 1255. 3. Schuurs, A. H. W. M., and Van Weemen, B. K. (1977) C/in. Chim. Acta 81, l-40. 4. Ishikawa, E. (1973) J. Biochem. 73, 1319-1321. 5. Kato, K., Hamaguchi, Y., Fukui, H., and Ishikawa, E. (1975) J. Biochem. 78, 235-237. 6. Kitagawa, T., and Aikawa, T. (1976) J. Biochem. 79, 233-236.
Tsuji, A., Maeda, M., Arakawa, H., and Matsuoka, K. (1976) Proc. Symp. Chem. Physiol. Pathol. 16, 47-51. 8. Numazawa, M., Haryu, A., Kurosaka, K., and Nambara, T. (1977) FEBS Lerf. 79, 396-398.
7.
AND TSUJI
18.
Arakawa, H., Maeda, M., and Tsuji, A. (1977) BNSKAK (Anal. Chem. Japan) 26, 322-327. Tsuji, A., Maeda, M., Arakawa, H., and Matsuoka, K. (1978) 10th Int. Congr. Clin. Chem., Mexico (Abstracts pp. 69). Comoglio, S., and Celada, F. (1976) J. lmmunol. Methods 10, 161-170. Ogihara, T., Miyai, K., Nishi, K., Ishibashi, K., and Kumahara, Y. (1977) J. Clin. Endocrinol. Metabol. 44, 91-95. Kobayashi, Y., Ogihara, T., Amitani, K., Watanabe, F., Kiguchi, T., Ninomiya, I., and Kumahara, Y. (1978) Steroids 32, 137- 144. Erlanger, B. F., Borek, F., Beiser, S. M., and Lieberman, S. (1967) Methods in Immunology and Immunochemistry (Williams, C. A., and Chase, M. C., eds.), Vol. 1, pp. 145-151, Academic Press, New York. Avrameas, S., and Guilbault, B. (1972) Biochimie 54, 837-842.
Glick, D. (1977) Clin. Chem. 23, 1465-1471. 20. Schroeder, H. R., Carrico, R. J., Boguslaski, R. C., and Christner, J. (1976) Anal. Biochem. 72, 19.
283-292.
21. Carrio, R. J., Yeung, K. K., Schroeder, H. R., Bogulaski, R. C., Buckler, R. T., and Christner, J. E. (1976) Anal. Biochem. 76, 95-110. 22. Puget, K., Michelson, A. M., and Avrameas, S. (1977) Anal. Biochem. 79, 447-456. 23. Van Weemen, B. K., and Schuurs, A. H. W. M. (1975) Immunochemistry 12, 667-670.
97, 248-254
BIOCHEMISTRY
Chemiluminescence
HIDETOSHI School
of Pharmaceutical
(1979)
Enzyme Immunoassay Peroxidase as Label ARAKAWA, Sciences,
Showa
MASAKO University,
MAEDA, Hatanodai,
of Cortisol
AND AKIO Shinagawa-ku.
Using
TSUJI Tokyo,
Japan
Received December 5, 1978 A new and highly sensitive enzyme immunoassay of cortisol was established using horseradish peroxidase as the label. Separation of free and bound cortisol was effected by insolubilized anti-cortisol antibody which was prepared by coupling the purified immunoglobulin G of antiserum with Sepharose 4B. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. Comparison of assay results obtained by radioimmunoassay and this enzyme immunoassay showed excellent agreement of results in all cases (r = 0.913). The detection limit of cortisol was about 1Opgper assay tube. This enzyme immunoassay,is applicable to the routine determination of plasma cortisol.
Since the first report by Van Weemen and Schuurs (l), a number of studies (2,3) have been reported on the enzyme immunoassay (EIA)’ for various hormones and drugs. EIAs, like radioimmunoassay (RIA), make use of conjugate to amplify the sensitivity of immunological detection methods. However, many of them are not as sensitive as the corresponding RIAs, because enzyme activities in EIAs are usually assayed colorimetrically. Fluorophotometric methods for the determination of enzyme activity have been employed in order to increase the sensitivities of EIAs using glucoamylase (4), p-D-galactosidase (5,6), and peroxidase (7,8). In this report, we have attempted to develop a highly sensitive and clinically useful EIA for cortisol in plasma using peroxidase as a label enzyme, based on chemiluminescence reaction. MATERIALS Reagents. Luminol, oxide, S-aminosalicylic
AND METHODS
30% hydrogen acid, tyramine,
perand
1 Abbreviations used: EIA, enzyme immunoassay; RIA, radioimmunoassay; BSA, bovine serum albumin: IgG, immunoglobulin G; HRP, horseradish peroxidase. 0003-2697/79/120248-07$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
248
cyanogen bromide were purchased from Tokyo Kasei Company (Tokyo). Sepharose 4B, Bio-Gel P 60, and DEAE-cellulose were products of Seikagakukogyo Company (Tokyo). Cortisol and various steroids were obtained from Sigma Chemical Company, and horseradish peroxidase (types III and VI) used as the label enzyme was also purchased from Sigma Chemical Company. Other reagents were of reagent grade. 6 /3Hemisuccinoxycortisol, cortisol 3-( Ocarboxymethyl)oxime, and antiserum (rabbit) against 6 rY-hemisuccinoxycortisolBSA were supplied from Dr. D. K. Fukushima, the Institute for Steroid Research and the Division of Oncology, Montefiore Hospital and Medical Center, New York. 21-Hemisuccinoxycortisol and cortisol 3-( 0-carboxymethyl)oxime were prepared according to the literature (9). The anti-cortisol sera against their BSA conjugates were prepared by the Central Research Institute of Tori Pharmaceutical Company and the Research Division of Teikokuzoki Company (Tokyo). The IgG fraction of antiserum was purified by DEAEcellulose column chromatography after precipitation with ammonium sulfate.
CHEMILUMINESCENCE
ENZYME
IMMUNOASSAY
OF CORTISOL
249
lene chloride for 1 min. A 50-~1 aliquot of antibody (imthe organic phase was transferred to a glass The insolubilized antibody of cortisol was prepared by the method of tube and dried under a stream of nitrogen, Afen et al. (10). Sepharose 4B (50 ml) was and the residue was dissolved by adding activated at pH 10.5 by mixing with 50 ml 0.1 ml of phosphate buffer containing 0.1% BSA. The resultant sample solution was subof a cyanogen bromide solution (50 mg/ml aqueous solution) for 7 min. After washing mitted to the enzyme immunoassay. with water and 0.1 M sodium bicarbonate solution, the activated Sepharose 4B was Enzyme Immunoassay Procedure mixed with 0.1 g purified anti-cortisol IgG (rabbit) in 10 ml of 0.1 M sodium bicarbonate Method A (jirst-antibody solid-phase of a sample solution. The reaction was run at room tem- method). One-tenth milliliter perature overnight with slow stirring. Then, solution or a cortisol standard solution conthe coupled product was washed by the taining 0 to 1 ng of cortisol was incubated method of Wide (11). The washed insoluovernight at 4°C with 0.5 ml of a diluted imsuspension (1:50) (insolubilized antibody was finally suspended in munosorbent bilized first antibody) on a immunorotor. buffer and stored at 4°C. The insolubilized second antibody was also prepared by the After addition of 0.5 ml of a diluted HRPsame procedure with anti-rabbit IgG frac- cortisol conjugate solution (1:l X 104-3 x 104), the mixture was again incubated for tion (goat). Preparation oj’ HRP-cortisol conjugate. 4 h at 4°C on a immunorotor. The incubation HRP-Cortisol conjugates were prepared by mixture was centrifuged at 3000 t-pm for 5 the mixed anhydride method from 2 l-hemimin. One milliliter of the supernatant was succinoxycortisol, cortisol 3-( O-carboxyaspirated off, and the precipitated immunosorbent in the tube was washed three times methyl)oxime, and 6-p-hemisuccinoxycortieach with 2 ml of saline. And then, the persol. For example, 1 mg of 21-hemisuccinoxyoxidase activity was measured by the chemicortisol was dissolved in 0.2 ml of dioxane, and then 10 ,ul of tri-n-butylamine and 4 ~1 luminescence (12), fluorophotometric (13), (1) method. of isobutylchlorocarbonate were added to it. or spectrophotometric Method B (double-antibody solid-phase The resultant solution was stirred for 30 min at 10°C and then mixed with cooled method). Each 0.1 ml of a diluted anti-cortisol 1 ml enzyme solution containing 15 mg of serum (1:500) and a cortisol standard soluhorseradish peroxidase. The mixture was tion or a sample solution was mixed and kept at pH 8.0-8.5 for 5 h in ice-water bath. incubated for 1 hat room temperature. After The resultant reaction mixture was dialyzed addition of 0.5 ml of a diluted insolubilized against 0.05 M phosphate buffer and then second-antibody suspension ( 1:50), the chromatographed on Bio-Gel P-60 column mixture was incubated overnight at 4°C equilibrated in 0.05 M phosphate buffer con- on a immunorotor. One-half milliliter of a taining 0.9% sodium chloride (pH 7.0). The HRP-cortisol conjugate solution (1: 1. x 104excluded peak fractions were diluted with 3 x 104) was then added to all tubes which 0.05 M phosphate buffer containing 0.1% were incubated for a further 1 h at 4°C. The BSA, and stored at 4°C. 6-@Hemisuccinoxyincubation mixture was centrifuged at 3000 cortisol and cortisol 3-( O-carboxymethyl)rpm for 5 min, and the supernatant was oxime were also conjugated with HRP by aspirated and discarded. The immunothe same procedure as described above. sorbent was washed three times each with Plasma extraction. Ten microliters of 2 ml of saline, and then submitted to plasma was diluted with 50 ~1 of distilled measurement of the peroxidase activity water, and extracted with 500 ~1 of methyby the chemiluminescence method.
Preparation munosorbent).
of insolubilized
250
ARAKAWA,
MAEDA,
Method C (double-antibody method). The assay was caried out in a similar manner to the method described by Van Weemen et al. (1).
AND TSUJI
methods. In this paper, HRP was selected for the label enzyme, because HRP is widely used in various EIAs and is one of the best studied enzymes, available cheaply, and very stable under assay and storage conditions. Several procedures for coupling enzyme to hormone or drug have been developed. In the case of steroids, the mixed anhydride method (17) and soluble carbodiimide method were used for preparation of conjugate. Both methods were examined in this paper, and the better conjugate was obtained by the mixed anhydride method. The highest active fraction obtained by a gel chromatographic purification could be used for the assay in 1 x 104- to 3 x 104fold dilution. The binding ratio of cortisol to HRP in the conjugate was determined to be about 2 to 1 by spectrophotometry. The total enzyme activity of fractions that could be used in EIA was approximately 43% of the initial total activity. The choice of the separation procedure is important to enhance the sensitivity in heterogeneous EIA. First-antibody solidphase method (method A), double-antibody solid-phase method (method B), and doubleantibody method (method C) were ex-
Measurement of peroxidase activity by chemiluminescence method (12). Each 0.5
ml of 0.01 M phosphate buffer (pH 7.0), 0.01 M H,Oz solution, and 0.01 M luminol solution was added to the washed immunosorbent in the test tube, respectively. After mixing well by a Vortex-type mixer for 5 s, the chemiluminescence intensity was measured for 10 s by a photon counter (HTV-C 767, Hamamatsu TV Co., Japan). RESULTS AND DISCUSSION
Although radioimmunoassay methods have been widely used for determination of steroid hormones in biological fluids, EIAs of steroids have recently been developed, because of the biohazard and problems of disposal resulting from the use of radioisotopes. EIAs of cortisol using /3-D-galactosidase (14) and alkaline phosphatase (15,16) as label enzyme have been reported. In those EIAs, the enzyme activities were assayed by calorimetric 100
100
iR
Y 5
5 x
X 50 2 t L*
Ino 2
4 ’ 0.01
0.025
I 0.05
I 0.10 cortiso1
I 0.25
I 0.50
I 1.0
L &tube
2
5o
I h
1,
' 0.25
1
I
I
0.5
1.0
1
2.5
I
I
5.0
10
n&tube
cortiso1
FIG. 1. Standard curves of cortisol by chemiluminescence enzyme immunoassay. (A) (0 --) First-antibody solid-phase method (method A). (- - 0 - -) Double-antibody solid-phase method (method B). (II, andB) Chemiluminescence intensities of HRP-cortisol conjugate bound to immunosorbent in the absence and presence of cortisol, respectively. (B) Double-antibody method (method C). (F, and F) Chemiluminescence intensities of the supernatants in the absence and presence of cortisol, respectively. (T) Chemiluminescence intensity of the supernatant in the absence of anti-cortisol serum and cortisol.
CHEMILUMINESCENCE
ENZYME
amined. The typical standard curves by these methods are shown in Fig. 1. Fig. 1A was obtained by plotting (BIB,,) x 100 against the amount of cortisol (B, and B: the chemiluminescence intensities by HRPcortisol conjugate bound to immunosorbent in the absence and the presence of cortisol, respectively). Fig. 1B was prepared by plotting (F-F,)I( T-F,,) x 100 against the amount of cortisol (F, and F: the chemiluminescence intensities of the supernatants in the absence and the presence of cortisol, respectively. T: the chemiluminescence intensity of the supernatant in the absence of anti-cortisol serum and cortisol). Van Weemen et al. (1) and Avrameas et al. (18) showed that the double-antibody solidphase method gave higher sensitivity, greater precision, and more rapid separation than the insolubilized first-antibody method in EIAs for human chorionic gonadotropin and IgG, respectively. However, in our study, the sensitivity of the double-antibody solid-phase method is almost same as that of the insolubilized first-antibody method. Therefore, the first-antibody solidphase prepared from anti-cortisol serum against 21-hemisuccinoxycortisol-BSA was used for the separation of bound and free fraction in the assay of plasma samples (method A) and the double-antibody solidphase method (method B) was used for the test of comparison of heterologous and homologous combinations of antibodies and HRP-cortisol conjugates. As shown in Fig. lA, cortisol could be determined in the range from 10 pg to 2.5 ng. The sensitivity of these methods are comparable to that of the radioimmunoassay. The repeatability, assessed by evaluating the coefficients of variation at three cortisol levels (SO, 100 and 500 pg), were 6.6, 7.5, and 10.4% with method A and 8.2, 3.9, and 7.8% with method B, respectively. The cross-reactivity of some steroids with this method was examined. Crossreactivity of 1 l-desoxycortisol, 17+hydroxyprogesterone, corticosterone, progester-
IMMUNOASSAY
OF CORTISOL
251
one, and dehydroepiandrosterone were 83.7,38.5, 18.9,7.1, and0.9%, respectively. These values were approximately equal to those obtained by RIA using the same anticortisol serum which was used in EIA. The percentage of cross-reaction of 1I-desoxycortisol and 17 a-hydroxyprogesterone was considerably high, but the concentrations of these steroids in plasma are insignificant relative to that of cortisol. Therefore, any separation step prior to EIA was unnecessary for the assay of human plasma samples. The label enzyme activity is usually measured by visible or ultraviolet spectrophotometry. Fluorophotometric methods have been used in highly sensitive assays for the labels, glucoamylase (4), and /3-D-galactosidase (5,6). The peroxidase activity in EIA for estradiol has been assayed by fluorophotometric method (8) using homovanillic acid and H,O, as substrate. Recently, chemiluminescence reaction of luminol is used for the determination of certain trace metals and other substances of biological interest (19). Schroeder et al. have developed the specific protein-binding reaction monitored using bioluminescence reaction with ligand-NAD (20) or ligand-ATP conjugate (21). Puget et al. (22) have also reported the chemiluminescence technique for the determination of peroxidase and its application to the estimation of antibody bound to mouse lympocytes using luminolH,O, or pholad luciferin as substrate. In our previous paper, we established the assay of peroxidase activity using luminolHzOz as substrate and preliminarily applied it to EIA (12). This method could be favorably applied to the EIA of cortisol. The detection limit of cortisol was approximately 10 pg per assay tube, as shown in Fig. IA, comparable to that of RIA and superior to those of the EIAs for cortisol reported by Comoglio (15), Ogihara (16), and Kobayashi (18). The simple comparisons between sensitivities of assays are difficult, because they are dependent on various factors, such as the affinity of antigen-antibody reaction
252
ARAKAWA,
MAEDA,
and the separation methods of bound and free fractions. Therefore, the comparison between chemiluminescence and spectrophotometric and fluorophotometric methods for the assay of enzyme activity was carried out under the same immune reaction conditions. As shown in Table 1, the chemiluminescence method was more sensitive than the spectrophotometric method with 5-aminosalicylic acid and the fluorophotometric method with tyramine. Moreover, the time for the assay of enzyme activity by the chemiluminescence method was only 10 s. On the other hand, it took 30 min or more to get the measurable optical intensity by spectrophotometry and fluorophotometry. In EIAs of steroid, the systems in which the same steroid derivative is used both for preparing the immunogen and for preparing the steroid-enzyme conjugate are referred to as “homologous” in contrast to “heterologous” systems, in which different steroid derivatives are used. Van Weemen et al. (23) suggested that the sensitivity of EIA for estrogen could be considerably improved when the heterologous systems were used. Recently, Numazawa et al. (8) reported a similar effect in the EIA of estradiol, while Ogihara et al. (16) reported that cortisol could not be assayed TABLE COMPARISON OF RESULTS SPECTROPHOTOMETRIC
Method Fluorophotometric
method
OBTAINED METHODS
AND TSUJI
by the heterologous system using 21-hemisuccinoxycortisol-alkaline phosphatase conjugate and anti-cortisol antiserum raised against cortisol3-(O-carboxymethyboximeBSA conjugate. In this paper, the homologous and heterologous systems were brought about by utilizing the different positions on cortisol nucleus, namely C-3, C-6, and C-2 1, through which enzyme and carrier protein were linked to cortisol, respectively. The concentrations of cortisol which replaced 50% of HRP-cortisol conjugate bound to insolubilized antibody by method B were determined as the sensitivities of various systems. The results are given in Table 2. As shown in Table 2, the highest sensitivity was obtained by the heterologous system combining anti-6a+hemisuccinoxycortisol-BSA with HRP conjugate of cortisol 3-( 0-carboxymethyl)oxime, of which the midpoint is 0.12 &assay tube. However, the detection limit obtained by this system was almost the same as that of the homologous system with 21-hemisuccinoxycortisol. The steepness of the assay curves varied from each other. From these results, it is difficult to reach a systematic conclusion on the effect of the combination of antibody and HRPcortisol conjugate being used to the sensitivity of EIAs for cortisol, because the 1
BY CHEMILUMINESCENCE, FOR DETERMINATION
FLUOROPHOTOMETIUC OF PEROXIDASE ACTIVITY~
Added
AND
Sensitivity
Reproducibility
(PP)
(CV%)b
25
2.8-6.8
100 250
100.6 89.2
bzhl)
Recovery (%I
Spectrophotometric
method
100
2.9-11.6
250 500
111.6 105.8
Chemiluminescence
method
10
5.2-9.3
250 500
85.2 83.2
a Method A of the enzyme immunoassay was used. “n=5.
CHEMILUMINESCENCE
ENZYME
IMMUNOASSAY
TABLE MIDPOINTS
2
FOR CORTISOL IN ENZYME IMMUNOASSAY COMPARED WITH CORRESPONDING
Cortisol derivative in immunogen
(METHOD HOMOLOGOUS
B) WITH SITE HETEROLOGY ASSAYS”
6ol-Hemisuccinoxy cortisol-BSA
2 1-Hemisuccinoxy cortisol-BSA
Cortisol derivative in HRP conjugate
253
OF CORTISOL
Dilution of antibody
Cortisol 3-(O-carboxymethyl) oxime-BSA
Dilution of antibody
Midpoint
21-Hemisuccinoxy cortisol-HRP
500
0.4 ng (X 10000)
10
50 ng (X 1000)
8ooo
0.5 ng (X 10ooo)
6@Hemisuccinoxy cortisol-HRP
50
50 ng ( x 2500)
500
0.5 ng ( x 10000)
500
>l.O /.&g ( x 5000)
Cortisol 3-(O-carboxymethyl) oxime-HRP
20
>l.O /.&g ( x 5000)
1000
0.12 ng (X30000)
500
>I.0 pg ( x 5000)
n Value in parentheses: dilution of HRP-cortisol pension used in this experiment: 50 fold.
Dilution of antibody
conjugate. Dilution of insolubilized
immune reactivity of antibody and cortisolenzyme conjugate have large individual differences. The cortisol levels in 28 plasma samples were determined by method A of EIA and RIA in order to assess the reliability of the present method. As illustrated in Fig. 2, the values obtained by EIA and RIA are in excellent agreement, the correlation co-
I
Midpoint
I
1
50
100
Midpoint
second-antibody sus-
efficient for cortisol samples in the range from 5 to 250 rig/ml of plasma being 0.913. The values obtained by EIA tended to be slightly higher than the corresponding values obtained by RIA: In conclusion, a highly sensitive and practically useful EIA for cortisol in plasma has been established. The chemiluminescence reaction using peroxidase-luminol-H,O,
I
Cortisol
(g/ml
Enzyme
hWlOaSSay
I
150 in plasma) (Method
200
250
A)
FIG. 2. Correlation between EIA and RIA values of cortisol in plasma.
254
ARAKAWA,
MAEDA,
system was most sensitive and rapid for the assay of enzyme activity. This system may be applicable extensively for the determination of various steroid hormones and drugs in the biological materials.
9. Tsuji, A., Smulowtiz, M., Liang, J. S. C., and Fukushima, D. K. (1974) Steroids 24,739-751. 10. Afne, R.. Porath, J., and Emback, S. (1967) Nature (London) 214, 1302- 1304. 11. Wide, L. (1970) Acta Endocrinol. (Suppl.) 142, 207-218. 12.
ACKNOWLEDGMENTS This work was supported in part by a Grant 357620 from the Ministry of Education of Japan. The authors are indebted to Dr. David K. Fukushima (the Institute for Steroid Research and the Division of Oncology, Montefiore Hospital and Medical Center, New York) for a gift of anti-cortisol serum and cortisol derivatives, and Mr. H. Tanaka (the Central Research Institute of Tori Pharmaceutical Co., Tokyo) for preparing anti-corns01 serum.
13. 14. IS. 16. 17.
REFERENCES 1. Van Weemen, B. K., and Schuurs, A. H. W. M. (1971) FEBS Let?. 15, 232-236. 2. Wisdom, G. B. (1976) C/in. C&m. 22, 1243- 1255. 3. Schuurs, A. H. W. M., and Van Weemen, B. K. (1977) C/in. Chim. Acta 81, l-40. 4. Ishikawa, E. (1973) J. Biochem. 73, 1319-1321. 5. Kato, K., Hamaguchi, Y., Fukui, H., and Ishikawa, E. (1975) J. Biochem. 78, 235-237. 6. Kitagawa, T., and Aikawa, T. (1976) J. Biochem. 79, 233-236.
Tsuji, A., Maeda, M., Arakawa, H., and Matsuoka, K. (1976) Proc. Symp. Chem. Physiol. Pathol. 16, 47-51. 8. Numazawa, M., Haryu, A., Kurosaka, K., and Nambara, T. (1977) FEBS Lerf. 79, 396-398.
7.
AND TSUJI
18.
Arakawa, H., Maeda, M., and Tsuji, A. (1977) BNSKAK (Anal. Chem. Japan) 26, 322-327. Tsuji, A., Maeda, M., Arakawa, H., and Matsuoka, K. (1978) 10th Int. Congr. Clin. Chem., Mexico (Abstracts pp. 69). Comoglio, S., and Celada, F. (1976) J. lmmunol. Methods 10, 161-170. Ogihara, T., Miyai, K., Nishi, K., Ishibashi, K., and Kumahara, Y. (1977) J. Clin. Endocrinol. Metabol. 44, 91-95. Kobayashi, Y., Ogihara, T., Amitani, K., Watanabe, F., Kiguchi, T., Ninomiya, I., and Kumahara, Y. (1978) Steroids 32, 137- 144. Erlanger, B. F., Borek, F., Beiser, S. M., and Lieberman, S. (1967) Methods in Immunology and Immunochemistry (Williams, C. A., and Chase, M. C., eds.), Vol. 1, pp. 145-151, Academic Press, New York. Avrameas, S., and Guilbault, B. (1972) Biochimie 54, 837-842.
Glick, D. (1977) Clin. Chem. 23, 1465-1471. 20. Schroeder, H. R., Carrico, R. J., Boguslaski, R. C., and Christner, J. (1976) Anal. Biochem. 72, 19.
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21. Carrio, R. J., Yeung, K. K., Schroeder, H. R., Bogulaski, R. C., Buckler, R. T., and Christner, J. E. (1976) Anal. Biochem. 76, 95-110. 22. Puget, K., Michelson, A. M., and Avrameas, S. (1977) Anal. Biochem. 79, 447-456. 23. Van Weemen, B. K., and Schuurs, A. H. W. M. (1975) Immunochemistry 12, 667-670.
